JP4847129B2 - Wound healing promoter - Google Patents
Wound healing promoter Download PDFInfo
- Publication number
- JP4847129B2 JP4847129B2 JP2005506851A JP2005506851A JP4847129B2 JP 4847129 B2 JP4847129 B2 JP 4847129B2 JP 2005506851 A JP2005506851 A JP 2005506851A JP 2005506851 A JP2005506851 A JP 2005506851A JP 4847129 B2 JP4847129 B2 JP 4847129B2
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- wound healing
- preparing
- sheet
- healing promoter
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Description
本発明は、創傷治癒を促進するための創傷治癒促進材、その調製方法、調製デバイス、およびそれを用いた創傷部位の再生促進方法に関する。 The present invention relates to a wound healing promoter for promoting wound healing, a preparation method thereof, a preparation device, and a method for promoting regeneration of a wound site using the same.
近年、創傷の治癒に関するメカニズムが次第に明らかになっており、その一つとして、白血球、血小板といった血液中の細胞が産生するさまざまな成長因子が組織の再生に重要な働きをすることが知られている。
ここでいう成長因子とは、血液凝固や炎症細胞の遊走を始めとして、線維芽細胞の増殖や細胞外マトリックスの合成、血管新生およびそれらの再構築に至るまでの創傷治癒の諸現象を複雑な細胞間ネットワーク関係のもとで直接的または間接的に制御するものである。例えば、血小板由来の成長因子のひとつとして発見されたＰＤＧＦ（血小板由来成長因子、ｐｌａｔｅｌｅｔ−ｄｅｒｉｖｅｄ ｇｒｏｗｔｈ ｆａｃｔｏｒ）は線維芽細胞や平滑筋細胞の遊走、増殖促進、単球・好中球の強力な走化性作用、さらには線維芽細胞のコラーゲン、コラゲナーゼ合成促進因子としての作用が明らかになっている。これについてＰｉｅｒｃｅらのグループは、ウサギの耳に軟骨まで達する傷をつくりＰＤＧＦ−ＢＢを投与したところ、ＰＤＧＦ−ＢＢを投与しない傷よりも速やかに損傷の修復が行われることを示している（Ｊ．Ｃｅｌｌ Ｂｉｏｃｈｅｍ．１９９１ ４５：３１９−３２６）。ＰＤＧＦはその名前が示すとおり血小板に大量に含まれるが、それ以外にマクロファージや血管内皮細胞、平滑筋細胞などの細胞でも産生されている。創傷治癒に関連する成長因子としては、ＰＤＧＦ−ＢＢの他にもＦＧＦ（線維芽細胞成長因子；ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ）、ＶＥＧＦ（血管内皮増殖因子；ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ）、ＴＧＦ−β（形質転換性成長因子；ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ）、ＥＧＦ（上皮成長因子；ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ）などの成長因子が明らかにされており、その一部は臨床応用されるようになってきている。
また、創傷治癒の別のメカニズムとして、血液細胞自身の創傷治癒への関与といった側面からも幾つかの知見が得られている。例えばＫａｌｋａらは、ヒト末梢血中の単核球細胞をｉｎ ｖｉｔｒｏで培養した後、血管狭窄処置を行ったヌードラットの虚血部位にその培養細胞を移植すると細胞が生着して新たに血管を生成し虚血部位を治癒することを示しており、末梢血由来の白血球は上述の成長因子産生以外にも血管新生に直接関与するという面からも創傷治癒効果が期待されている（ＰＮＡＳ．２０００ ９７：３４２２−３４２７）。さらに、ヒト末梢血中の単球分画をｉｎ ｖｉｔｒｏでさまざまな成長因子を用いて培養することにより上皮細胞、血管内皮細胞、肝細胞、そして神経細胞といった細胞に分化する方法もＺｈａｏらによって示されており、細胞と成長因子の組合せがさらなる創傷治癒効果を示すことも期待される（ＰＮＡＳ．２００３ １００：２４２６−２４３１）。
創傷の治癒に際しては、特定の成長因子や細胞を患者に投与するが、その手法は主に全身投与または局所投与に二分される。しかしながら全身投与は、特に成長因子においては、投与量が多すぎる場合には例えばＶＥＧＦでは低血圧を、ｂＦＧＦでは腎毒性を引き起こす危険性がそれぞれ示唆されており、投与量の決定には極めて慎重な判断が必要であった。一方、局所投与は成長因子や細胞の何れにも適していると思われるが、局所での成長因子濃度を一定期間保持するのは困難である。また、創傷治癒における細胞同士の情報伝達は上に挙げたような多種の因子の作用する複雑な系であるから、単独の成長因子の投与では治癒効果は不十分な場合が多く、創傷治癒に関連する多種の成長因子の投与が必要であると考えられている。なお、局所投与を実現する別法としては遺伝子治療が考えられるが、遺伝子治療は現在まだ安全性が十分に確立されていない。
その点、成長因子に比べて特定細胞の局所投与は有効性が高いと考えられ、様々な技術検討がなされている。例えば、遠心分離により成長因子を産生する血小板を血液から濃縮し、これにフィブリノーゲンとトロンビンを加えて血小板糊を調製し、創傷部位に塗布する方法がある（Ｉｎｔ．Ｊ．Ａｒｔｉｆ．Ｏｒｇａｎｓ．２００２ ２５（４）：３３４−８）。しかし、この方法では調製に時間と手間がかかり、また、糊の機械的強度が低いという問題があった。特開２００１−２０４８０７号公報には、細胞を含むゲルの機械的強度を強くするために、高分子材料成型物からなる骨格の内部に線維芽細胞等の細胞を含むゲルを形成させた医用材料が開示されている。ところが、この材料は人工皮膚を主な用途とするものであり、白血球、血小板等をこのゲルの中に含んでいないことから創傷治癒促進の効果は期待できなかった。またゲル中の細胞は、ゲル内に固定されているため細胞が何らかの因子を産生したとしても、その因子が創傷部位に到達するのに時間を要するという問題もあった。また、米国特許第６０４９０２６号明細書には、移植片として骨髄中の結合組織前駆細胞を基体（ｓｕｂｓｔｒａｔｅ）表面に付着させる為のキットが提案されており、移植片の成長を促す為に血小板を更に付着させることが記載されている。しかしながらこれは主に骨の移植片に関する発明であり、創傷の治癒促進、すなわち生体側創傷部位の細胞の成長を促進することを目的としたものではなかった。従って、白血球を移植片に付着させるという開示は一切されていない。
以上述べたとおり、創傷治癒分野おいては、創傷治癒効果のある血液細胞をより効率的に利用した創傷治癒（促進）材については知られておらず、そのような技術革新が望まれていた。In recent years, mechanisms related to wound healing have become increasingly clear, and one of them is known that various growth factors produced by blood cells such as leukocytes and platelets play an important role in tissue regeneration. Yes.
The growth factor here refers to complex phenomena of wound healing from blood coagulation and migration of inflammatory cells to fibroblast proliferation, extracellular matrix synthesis, angiogenesis and their remodeling. It is controlled directly or indirectly under the network relationship between cells. For example, PDGF (platelet-derived growth factor), which was discovered as one of platelet-derived growth factors, is a fibroblast and smooth muscle cell migration, proliferation promotion, and a powerful monocyte / neutrophil migration. It has been clarified that it acts as a factor for promoting the synthesis of collagen and collagenase in fibroblasts. In this regard, the group of Pierce et al. Has shown that wounds reaching the cartilage in the ears of rabbits and PDGF-BB is administered, repairing the damage more quickly than wounds not administered PDGF-BB (J Cell Biochem. 1991 45: 319-326). As its name suggests, PDGF is contained in large amounts in platelets, but it is also produced in cells such as macrophages, vascular endothelial cells, and smooth muscle cells. Growth factors related to wound healing include FGF (fibroblast growth factor), VEGF (vascular endothelial growth factor), TGF-β (transformability) in addition to PDGF-BB. Growth factors such as growth factor (transforming growth factor) and EGF (epidermal growth factor) have been clarified, and some of them have been clinically applied.
In addition, as another mechanism of wound healing, some findings have been obtained from the aspect of the involvement of blood cells themselves in wound healing. Kalka et al., For example, cultured mononuclear cells in human peripheral blood in vitro, and then transplanted the cultured cells into the ischemic site of a nude rat that had undergone vascular stenosis, resulting in new engraftment of the cells. In addition to the growth factor production described above, leukocytes derived from peripheral blood are expected to have a wound healing effect in terms of being directly involved in angiogenesis (PNAS. 2000 97: 3422-3427). Furthermore, Zhao et al. Also showed a method of differentiating monocyte fractions in human peripheral blood into cells such as epithelial cells, vascular endothelial cells, hepatocytes, and nerve cells by culturing in vitro with various growth factors. It is also expected that the combination of cells and growth factors will show an additional wound healing effect (PNAS. 2003 100: 2426-2431).
In wound healing, specific growth factors and cells are administered to a patient, and the procedure is mainly divided into systemic administration and local administration. However, systemic administration, especially in the case of growth factors, suggests that there is a risk of causing hypotension in VEGF, for example, in VEGF, and nephrotoxicity in bFGF. Judgment was necessary. On the other hand, local administration seems to be suitable for both growth factors and cells, but it is difficult to maintain a local growth factor concentration for a certain period. In addition, cell-to-cell information transmission in wound healing is a complex system in which various factors such as those listed above act. Therefore, the administration of a single growth factor is often inadequate for the healing effect. It is believed that administration of a variety of related growth factors is necessary. In addition, gene therapy can be considered as another method for realizing local administration, but safety of gene therapy has not yet been sufficiently established.
In that regard, local administration of specific cells is considered to be more effective than growth factors, and various technical studies have been conducted. For example, there is a method of concentrating platelets producing growth factors from blood by centrifugation, adding fibrinogen and thrombin to this to prepare platelet glue, and applying it to the wound site (Int. J. Artif. Organs. 2002 25 (4): 334-8). However, this method has a problem that it takes time and labor to prepare, and the mechanical strength of the glue is low. Japanese Patent Application Laid-Open No. 2001-204807 discloses a medical material in which a gel containing cells such as fibroblasts is formed inside a skeleton made of a molded polymer material in order to increase the mechanical strength of the gel containing cells. Is disclosed. However, this material is mainly used for artificial skin, and since the gel does not contain leukocytes, platelets, etc., the effect of promoting wound healing could not be expected. Further, since the cells in the gel are fixed in the gel, even if the cells produce some factor, there is a problem that it takes time for the factor to reach the wound site. In US Pat. No. 6,049,026, a kit for attaching connective tissue progenitor cells in bone marrow to the surface of a substrate as a graft is proposed, and platelets are used to promote the growth of the graft. Further, it is described that it is adhered. However, this is mainly an invention related to bone grafts, and was not intended to promote wound healing, that is, promote cell growth at the living wound site. Accordingly, there is no disclosure of attaching leukocytes to the graft.
As described above, in the field of wound healing, there is no known wound healing (promoting) material that efficiently uses blood cells with wound healing effects, and such technological innovation has been desired. .
本発明は上記した従来技術の問題点を解消することを解決すべき課題とする。即ち、本発明は、創傷治癒に効果のある細胞を短時間で効率よく濃縮し、創傷治癒の促進材として活用するための手段を提供することを解決すべき課題とした。
本発明者らは上記課題を解決するために鋭意検討した結果、表面に白血球および／または血小板を存在させたシート状多孔質材料が線維芽細胞の増殖を向上させ、創傷治癒関連の成長因子を産生することにより創傷治癒を促進することを見出し、本発明を完成するに至った。
即ち、本発明は以下に関する。
（１）少なくとも白血球および／または血小板が表面に存在するシート状多孔質体からなる創傷治癒促進材。
（２）シート状多孔質体を含む創傷治癒促進材であって、細胞増殖能を有する創傷治癒促進材。
（３）線維芽細胞の増殖能を有する（２）に記載の創傷治癒促進材。
（４）細胞増殖能がシート状多孔質体表面に存在する白血球および／または血小板に由来する（３）に記載の創傷治癒促進材。
（５）シート状多孔質体を含む創傷治癒促進材であって、成長因子産生能を有する創傷治癒促進材。
（６）対照血漿に対する成長因子の産生能が、血管内皮増殖因子（ＶＥＧＦ）の場合は５倍以上、血小板由来増殖因子−ＡＢ（ＰＤＧＦ−ＡＢ）の場合は２倍以上、形質転換性成長因子（ＴＧＦ−β１）の場合は２倍以上の何れかを満たす（５）に記載の創傷治癒促進材。
（７）成長因子の産生能が、シート状多孔質体表面に存在する白血球およびまたは血小板に由来する（６）に記載の創傷治癒促進材。
（８）シート状多孔質体の厚さが０．０１〜３ｍｍである（１）〜（７）の何れかに記載の創傷治癒促進材。
（９）シート状多孔質体が創傷部位の形状に合わせて変形可能である（１）〜（８）の何れかに記載の創傷治癒促進材。
（１０）シート状多孔質体が不織布である（９）に記載の創傷治癒促進材。
（１１）不織布の繊維径が０．３〜５０μｍ、嵩密度が０．０５〜０．５ｇ／ｃｍ３である（１０）に記載の創傷治癒促進材。
（１２）シート状多孔質体がスポンジ状構造体である（９）に記載の創傷治癒促進材。
（１３）スポンジ状構造体の平均孔径が１．０〜４０μｍである（１２）に記載の創傷治癒促進材。
（１４）シート状多孔質体の材質が天然高分子または合成高分子である（１）〜（１３）の何れかに記載の創傷治癒促進材。
（１５）シート状多孔質体の材質が疎水性高分子を主要成分とする合成高分子である（１４）に記載の創傷治癒促進材。
（１６）シート状多孔質体の材質が生分解性素材である（１３）または（１４）に記載の創傷治癒促進材。
（１７）白血球および／または血小板が末梢血、骨髄液または臍帯血由来である（１）〜（１６）の何れかに記載の創傷治癒促進材。
（１８）白血球および／または血小板が成熟細胞である（１）〜（１６）の何れかに記載の創傷治癒促進材。
（１９）白血球および／または血小板が自己由来である（１７）または（１８）に記載の創傷治癒促進材。
（２０）シート状多孔質体における白血球密度が６．０×１０６個／ｃｍ３以上および／または血小板密度が２．５×１０８個／ｃｍ３以上である（１）〜（１９）の何れかに記載の創傷治癒促進材。
（２１）シート状多孔質体に線維芽細胞を組み込んだ（１）〜（２０）の何れかに記載の創傷治癒促進材。
（２２）線維芽細胞が創傷組織と同じ組織由来の線維芽細胞である（２１）に記載の創傷治癒促進材。
（２３）シート状多孔質体がフィブリンを含む（１）〜（２２）の何れかに記載の創傷治癒促進材。
（２４）シート状多孔質体に少なくとも白血球および／または血小板を捕捉する工程を含む創傷治癒促進材の調製方法。
（２５）シート状多孔質体の厚さが０．０１〜３ｍｍである（２４）に記載の創傷治癒促進材の調製方法。
（２６）シート状多孔質体が不織布である（２４）または（２５）の何れかに記載の創傷治癒促進材の調製方法。
（２７）不織布の繊維径が０．３〜５０μｍ、嵩密度が０．０５〜０．５ｇ／ｃｍ３である（２６）に記載の創傷治癒促進材の調製方法。
（２８）シート状多孔質体がスポンジ状構造体である（２４）または（２５）に記載の創傷治癒促進材の調製方法。
（２９）スポンジ状構造体の平均孔径が１．０〜４０μｍである（２８）に記載の創傷治癒促進材の調製方法。
（３０）シート状多孔質体が血液細胞の選択分離性を有する（２４）〜（２９）の何れかに記載の創傷治癒促進材の調製方法。
（３１）シート状多孔質体の表面が血液細胞の選択分離性を有する（３０）に記載の創傷治癒促進材の調製方法。
（３２）シート状多孔質体が赤血球に比し白血球および／または血小板を選択的に捕捉するものである（３１）に記載の創傷治癒促進材の調製方法。
（３３）シート状多孔質体の白血球捕捉率が５０％以上および／または血小板捕捉率が５０％以上である（３２）に記載の創傷治癒促進材の調製方法。
（３４）少なくとも白血球および／または血小板を含む細胞浮遊液を濾過によりシート状多孔質体に捕捉する（２４）〜（３３）の何れかに記載の創傷治癒促進材の調製方法。
（３５）濾過がワンスルーにより行なわれる（３４）に記載の創傷治癒促進材の調製方法。
（３６）細胞浮遊液の濾過液量が４００ｍｌ未満である（３５）に記載の創傷治癒促進材の調製方法。
（３７）シート状多孔質体の有効濾過面積に対する濾過液量の比が１０ｍｌ／ｃｍ２未満である（３５）〜（３７）の何れかに記載の創傷治癒促進材の調製方法。
（３８）シート状多孔質体の容積に対する濾過液量の比が１００未満である（３５）〜（３７）の何れかにに記載の創傷治癒促進材の調製方法。
（３９）シート状多孔質体に対して細胞浮遊液を一回濾過させる（３５）〜（３８）の何れかに記載の創傷治癒促進材の調製方法。
（４０）シート状多孔質体に対する細胞浮遊液の濾過方向が一方向である（３５）〜（４０）の何れかに記載の創傷治癒促進材の調製方法。
（４１）濾過時間が２０分以内である（３５）〜（４０）の何れかに記載の創傷治癒促進材の調製方法。
（４２）濾過が体外循環により行なわれる（３４）に記載の創傷治癒促進材の調製方法。
（４３）濾過流速が２０〜２００ｍｌ／ｍｉｎである（４２）に記載の創傷治癒促進材の調製方法。
（４４）濾過時間が１０〜３００分である（４２）または（４３）に記載の創傷治癒促進材の調製方法。
（４５）細胞浮遊液が新鮮血である（２４）〜（４４）の何れかに記載の創傷治癒促進材の調製方法。
（４６）細胞浮遊液が採血後４８時間以内の新鮮血である（４５）に記載の創傷治癒促進材の調製方法。
（４７）細胞浮遊液が成熟細胞を主に含む（２４）〜（４６）の何れかに記載の創傷治癒促進材の調製方法。
（４８）細胞浮遊液が自己血由来である（２４）〜（４７）の何れかに記載の創傷治癒促進材の調製方法。
（４９）細胞浮遊液がクエン酸塩、ヘパリン類または水解酵素阻害剤の何れかを抗凝固剤として含む（２４）〜（４８）の何れかに記載の創傷治癒促進材の調製方法。
（５０）少なくとも白血球および／または血小板を捕捉したシート状多孔質体を培養する工程をさらに含む（２４）〜（４９）の何れかに記載の創傷治癒促進材の調製方法。
（５１）培養時に細胞活性化材を添加する（５０）に記載の創傷治癒促進材の調製方法。
（５２）シート状多孔質体に線維芽細胞を組み込む工程をさらに含む（２４）〜（５１）の何れかに記載の創傷治癒促進材の調製方法。
（５３）線維芽細胞をシート状多孔質体に接触させる、または線維芽細胞を細胞浮遊液に混合して濾過することにより組み込む（５２）に記載の創傷治癒促進材の調製方法。
（５４）線維芽細胞が創傷組織と同じ組織由来の線維芽細胞である（５３）に記載の創傷治癒促進材の調製方法。
（５５）シート状多孔質体にフィブリンを含ませる工程をさらに含む（２４）〜（５４）の何れかに記載の創傷治癒促進材の調製方法。
（５６）フィブリンが製剤由来である（５５）に記載の創傷治癒促進材の調製方法。
（５７）フィブリンが細胞浮遊液をシート状多孔質体に濾過した際のドレインを回収し濃縮して得たものである（５６）に記載の創傷治癒促進材の調製方法。
（５８）少なくとも白血球および／または血小板を捕捉後にシート状多孔質体を洗浄する工程をさらに含む（２４）〜（５７）の何れかに記載の創傷治癒促進材の調製方法。
（５９）少なくとも白血球および／または血小板をシート状多孔質体に捕捉する工程および／または該シート状多孔質体を洗浄する工程が、液体の入口と出口が付いた開放可能な液密容器内で行われる（２４）〜（５８）の何れかに記載の創傷治癒促進材の調製方法。
（６０）開放可能な液密容器からシート状多孔質体を取り出し、保存する工程をさらに含む（５９）に記載の創傷治癒促進材の調製方法。
（６１）（２４）〜（６０）の何れかに記載の創傷治癒促進材の調製方法により得られる創傷治癒促進材。
（６２）液体の注入と排出のための入口と出口が付いた開放可能な液密容器の内部に、容器内部を二室に隔絶し、一方を入口側に他方を出口側に通じるようにシート状多孔質体を配置した創傷治癒促進材の調製デバイス。
（６３）可撓性樹脂シートにシート状多孔質体を挟み込んで溶着または接着した軟質デバイスであり、可撓性樹脂シート部を剥がすことにより内部のシート状多孔質体を露出または取り出し可能とした（６２）に記載の創傷治癒促進材の調製デバイス。
（６４）平板状である（６３）に記載の創傷治癒促進材の調製デバイス。
（６５）締結手段を備えた硬質デバイスであり、該締結手段を解除することにより内部のシート状多孔質体を露出または取り出し可能とした（６２）に記載の創傷治癒促進材の調製デバイス。
（６６）円筒状である（６５）に記載の創傷治癒促進材の調製デバイス。
（６７）シート状多孔質体が反物状に巻かれて収容されている（６６）に記載の創傷治癒促進材の調製デバイス。
（６８）シート状多孔質体の厚さが０．０１〜３ｍｍである（６２）〜（６７）の何れかに記載の創傷治癒促進材の調製デバイス。
（６９）シート状多孔質体が不織布である（６２）〜（６８）の何れかに記載の創傷治癒促進材の調製デバイス。
（７０）不織布の繊維径が０．３〜５０μｍ、嵩密度が０．０５〜０．５ｇ／ｃｍ３である（６９）に記載の創傷治癒促進材の調製デバイス。
（７１）シート状多孔質体がスポンジ状構造体である（６２）〜（６８）の何れかに記載の創傷治癒促進材の調製デバイス。
（７２）スポンジ状構造体の平均孔径が１．０〜４０μｍである（７１）に記載の創傷治癒促進材の調製デバイス。
（７３）シート状多孔質体が血液細胞の選択分離性を有する（６２）〜（７２）の何れかに記載の創傷治癒促進材の調製デバイス。
（７４）シート状多孔質体の表面が血液細胞の選択分離性を有する（７３）に記載の創傷治癒促進材の調製デバイス。
（７５）シート状多孔質体が赤血球に比し白血球および／または血小板を選択的に捕捉するものである（７４）に記載の創傷治癒促進材の調製デバイス。
（７６）シート状多孔質体の白血球捕捉率が５０％以上および／または血小板捕捉率が５０％以上である（７５）に記載の創傷治癒促進材の調製デバイス。
（７７）容器の入口側および／または出口側にバッグを接続可能な接続部品を備えた（６２）〜（７６）の何れかに記載の創傷治癒促進材の調製デバイス。
（７８）容器の入口側に採血バッグおよび／または出口側に遠心バッグを備えた（７７）に記載の創傷治癒促進材の調製デバイス。
（７９）容器の入口側および出口側に体外循環回路を備えた（６２）〜（７６）の何れかに記載の創傷治癒促進材の調製デバイス。
（８０）滅菌袋に包装されて滅菌された（６２）〜（７９）の何れかに記載の創傷治癒促進材の調製デバイス。
（８１）容器の入口出口が外部に連通して包装、装置全体が滅菌袋に包装されて滅菌された（７７）に記載の創傷治癒促進材の調製デバイス。
（８２）（６２）〜（８１）の何れかに記載の創傷治癒促進材の調製デバイスを用いて得られる創傷治癒促進材。
（８３）（１）〜（２３）、（６１）および（８２）の何れかに記載の創傷治癒促進材を創傷部位に貼り付ける創傷部位の治療方法。
（８４）容器を開放し、シート状多孔質体の一面を容器から露出させた状態で貼り付ける（８３）に記載の創傷部位の治療方法。
（８５）容器からシート状多孔質体を取り出して貼り付ける（８４）に記載の創傷部位の治療方法。
（８６）創傷治癒促進材を調製後３０分以内に創傷部位に貼り付ける（８３）〜（８５）の何れかに記載の創傷部位の治療方法。
（８７）創傷が体表面である（８３）〜（８６）の何れかに記載の創傷部位の治療方法。
（８８）貼り付けた創傷治癒促進材を保護材により被覆固定する（８３）〜（８７）の何れかに記載の創傷部位の治療方法。
（８９）保護材が透水性を有さない素材からなるシートである（８８）に記載の創傷部位の治療方法。
（９０）保護材がガス透過性を有しかつ透水性を有さない素材からなるシートである（８９）に記載の創傷部位の治療方法。An object of the present invention is to solve the above-described problems of the prior art. That is, this invention made it the subject which should be solved to provide the means for efficiently concentrating the cell effective in wound healing in a short time, and utilizing as a wound healing promoter.
As a result of intensive studies to solve the above problems, the present inventors have found that a sheet-like porous material having leukocytes and / or platelets on the surface improves the proliferation of fibroblasts, and a growth factor related to wound healing. It has been found that it promotes wound healing by producing it, and the present invention has been completed.
That is, the present invention relates to the following.
(1) A wound healing promoter comprising a sheet-like porous body having at least leukocytes and / or platelets on the surface.
(2) A wound healing promoter comprising a sheet-like porous body, which has a cell growth ability.
(3) The wound healing promoter according to (2), which has the ability to proliferate fibroblasts.
(4) The wound healing promoter according to (3), wherein the cell proliferation ability is derived from leukocytes and / or platelets present on the surface of the sheet-like porous body.
(5) A wound healing promoter comprising a sheet-like porous body, which has a growth factor-producing ability.
(6) The growth factor production ability relative to the control plasma is 5 times or more in the case of vascular endothelial growth factor (VEGF), 2 times or more in the case of platelet-derived growth factor-AB (PDGF-AB), transforming growth factor In the case of (TGF-β1), the wound healing promoter according to (5), which satisfies at least two times.
(7) The wound healing promoter according to (6), wherein the growth factor-producing ability is derived from leukocytes and / or platelets present on the surface of the sheet-like porous body.
(8) The wound healing promoter according to any one of (1) to (7), wherein the sheet-like porous body has a thickness of 0.01 to 3 mm.
(9) The wound healing promoter according to any one of (1) to (8), wherein the sheet-like porous body is deformable according to the shape of the wound site.
(10) The wound healing promoter according to (9), wherein the sheet-like porous body is a nonwoven fabric.
(11) The wound healing promoter according to (10), wherein the nonwoven fabric has a fiber diameter of 0.3 to 50 μm and a bulk density of 0.05 to 0.5 g / cm 3 .
(12) The wound healing promoter according to (9), wherein the sheet-like porous body is a sponge-like structure.
(13) The wound healing promoter according to (12), wherein the sponge structure has an average pore size of 1.0 to 40 μm.
(14) The wound healing promoter according to any one of (1) to (13), wherein the material of the sheet-like porous body is a natural polymer or a synthetic polymer.
(15) The wound healing promoter according to (14), wherein the material of the sheet-like porous body is a synthetic polymer having a hydrophobic polymer as a main component.
(16) The wound healing promoter according to (13) or (14), wherein the material of the sheet-like porous body is a biodegradable material.
(17) The wound healing promoter according to any one of (1) to (16), wherein the white blood cells and / or platelets are derived from peripheral blood, bone marrow fluid or umbilical cord blood.
(18) The wound healing promoter according to any one of (1) to (16), wherein the white blood cells and / or platelets are mature cells.
(19) The wound healing promoter according to (17) or (18), wherein leukocytes and / or platelets are self-derived.
(20) The leukocyte density in the sheet-like porous body is 6.0 × 10 6 cells / cm 3 or more and / or the platelet density is 2.5 × 10 8 cells / cm 3 or more. The wound healing promoter according to any one of the above.
(21) The wound healing promoter according to any one of (1) to (20), wherein fibroblasts are incorporated into a sheet-like porous body.
(22) The wound healing promoter according to (21), wherein the fibroblast is a fibroblast derived from the same tissue as the wound tissue.
(23) The wound healing promoter according to any one of (1) to (22), wherein the sheet-like porous body contains fibrin.
(24) A method for preparing a wound healing promoter comprising a step of capturing at least leukocytes and / or platelets in a sheet-like porous body.
(25) The method for preparing a wound healing promoter according to (24), wherein the thickness of the sheet-like porous body is 0.01 to 3 mm.
(26) The method for preparing a wound healing promoter according to any one of (24) or (25), wherein the sheet-like porous body is a nonwoven fabric.
(27) The method for preparing a wound healing promoter according to (26), wherein the nonwoven fabric has a fiber diameter of 0.3 to 50 µm and a bulk density of 0.05 to 0.5 g / cm 3 .
(28) The method for preparing a wound healing promoter according to (24) or (25), wherein the sheet-like porous body is a sponge-like structure.
(29) The method for preparing a wound healing promoter according to (28), wherein the average pore size of the sponge-like structure is 1.0 to 40 μm.
(30) The method for preparing a wound healing promoter according to any one of (24) to (29), wherein the sheet-like porous material has selective separation of blood cells.
(31) The method for preparing a wound healing promoter according to (30), wherein the surface of the sheet-like porous body has selective separation of blood cells.
(32) The method for preparing a wound healing promoter according to (31), wherein the sheet-like porous body selectively captures leukocytes and / or platelets as compared with erythrocytes.
(33) The method for preparing a wound healing promoter according to (32), wherein the sheet-shaped porous body has a leukocyte capture rate of 50% or more and / or a platelet capture rate of 50% or more.
(34) The method for preparing a wound healing promoter according to any one of (24) to (33), wherein a cell suspension containing at least leukocytes and / or platelets is trapped in a sheet-like porous body by filtration.
(35) The method for preparing a wound healing promoter according to (34), wherein the filtration is performed by one-through.
(36) The method for preparing a wound healing promoter according to (35), wherein the amount of filtrate of the cell suspension is less than 400 ml.
(37) The method for preparing a wound healing promoter according to any one of (35) to (37), wherein the ratio of the amount of filtrate to the effective filtration area of the sheet-like porous body is less than 10 ml / cm 2 .
(38) The method for preparing a wound healing promoter according to any one of (35) to (37), wherein the ratio of the filtrate amount to the volume of the sheet-like porous body is less than 100.
(39) The method for preparing a wound healing promoter according to any one of (35) to (38), wherein the cell suspension is filtered once with respect to the sheet-like porous body.
(40) The method for preparing a wound healing promoter according to any one of (35) to (40), wherein the filtration direction of the cell suspension with respect to the sheet-like porous body is one direction.
(41) The method for preparing a wound healing promoter according to any one of (35) to (40), wherein the filtration time is within 20 minutes.
(42) The method for preparing a wound healing promoter according to (34), wherein the filtration is performed by extracorporeal circulation.
(43) The method for preparing a wound healing promoter according to (42), wherein the filtration flow rate is 20 to 200 ml / min.
(44) The method for preparing a wound healing promoter according to (42) or (43), wherein the filtration time is 10 to 300 minutes.
(45) The method for preparing a wound healing promoter according to any one of (24) to (44), wherein the cell suspension is fresh blood.
(46) The method for preparing a wound healing promoter according to (45), wherein the cell suspension is fresh blood within 48 hours after blood collection.
(47) The method for preparing a wound healing promoter according to any one of (24) to (46), wherein the cell suspension mainly contains mature cells.
(48) The method for preparing a wound healing promoter according to any one of (24) to (47), wherein the cell suspension is derived from autologous blood.
(49) The method for preparing a wound healing promoter according to any one of (24) to (48), wherein the cell suspension contains any one of citrate, heparins or a hydrolase inhibitor as an anticoagulant.
(50) The method for preparing a wound healing promoter according to any one of (24) to (49), further comprising a step of culturing a sheet-like porous body capturing at least leukocytes and / or platelets.
(51) The method for preparing a wound healing promoter according to (50), wherein a cell activating material is added during culture.
(52) The method for preparing a wound healing promoter according to any one of (24) to (51), further comprising a step of incorporating fibroblasts into the sheet-like porous body.
(53) The method for preparing a wound healing promoter according to (52), wherein fibroblasts are brought into contact with the sheet-shaped porous body, or incorporated by filtering fibroblasts into a cell suspension.
(54) The method for preparing a wound healing promoter according to (53), wherein the fibroblast is a fibroblast derived from the same tissue as the wound tissue.
(55) The method for preparing a wound healing promoter according to any one of (24) to (54), further comprising a step of including fibrin in the sheet-like porous body.
(56) The method for preparing a wound healing promoter according to (55), wherein fibrin is derived from the preparation.
(57) The method for preparing a wound healing promoter according to (56), wherein the fibrin is obtained by collecting and concentrating the drain when the cell suspension is filtered into a sheet-like porous body.
(58) The method for preparing a wound healing promoter according to any one of (24) to (57), further comprising a step of washing the sheet-like porous body after capturing at least leukocytes and / or platelets.
(59) The step of capturing at least leukocytes and / or platelets in the sheet-like porous body and / or washing the sheet-like porous body is performed in an openable liquid-tight container having a liquid inlet and outlet. The method for preparing a wound healing promoter according to any one of (24) to (58).
(60) The method for preparing a wound healing promoter according to (59), further comprising a step of taking out and storing the sheet-like porous body from an openable liquid-tight container.
(61) A wound healing promoter obtained by the method for preparing a wound healing promoter according to any one of (24) to (60).
(62) A sheet that separates the interior of the container into two chambers inside the openable liquid-tight container having an inlet and an outlet for injecting and discharging the liquid, and leads one to the inlet side and the other to the outlet side. Device for preparing a wound healing material in which a porous material is disposed.
(63) A soft device in which a sheet-like porous body is sandwiched and adhered to a flexible resin sheet, and the internal sheet-like porous body can be exposed or taken out by peeling off the flexible resin sheet portion. (62) The device for preparing a wound healing promoter according to (62).
(64) The device for preparing a wound healing promoter according to (63), which is flat.
(65) The device for preparing a wound healing promoter according to (62), wherein the device is a hard device including fastening means, and the internal sheet-like porous body can be exposed or removed by releasing the fastening means.
(66) The device for preparing a wound healing promoter according to (65), which is cylindrical.
(67) The preparation device for a wound healing promoter according to (66), wherein the sheet-like porous material is wound and accommodated in a fabric shape.
(68) The device for preparing a wound healing promoter according to any one of (62) to (67), wherein the thickness of the sheet-like porous body is 0.01 to 3 mm.
(69) The device for preparing a wound healing promoter according to any one of (62) to (68), wherein the sheet-like porous body is a nonwoven fabric.
(70) The device for preparing a wound healing promoter according to (69), wherein the nonwoven fabric has a fiber diameter of 0.3 to 50 µm and a bulk density of 0.05 to 0.5 g / cm 3 .
(71) The device for preparing a wound healing promoter according to any one of (62) to (68), wherein the sheet-like porous body is a sponge-like structure.
(72) The preparation device for a wound healing promoter according to (71), wherein the average pore size of the sponge-like structure is 1.0 to 40 μm.
(73) The preparation device for a wound healing promoter according to any one of (62) to (72), wherein the sheet-shaped porous body has selective separation of blood cells.
(74) The device for preparing a wound healing promoter according to (73), wherein the surface of the sheet-shaped porous body has selective separation of blood cells.
(75) The device for preparing a wound healing promoter according to (74), wherein the sheet-like porous body selectively captures leukocytes and / or platelets as compared with erythrocytes.
(76) The device for preparing a wound healing promoter according to (75), wherein the sheet-like porous body has a leukocyte capture rate of 50% or more and / or a platelet capture rate of 50% or more.
(77) The device for preparing a wound healing promoter according to any one of (62) to (76), comprising a connecting part capable of connecting a bag to the inlet side and / or the outlet side of the container.
(78) The device for preparing a wound healing promoter according to (77), comprising a blood collection bag on the inlet side of the container and / or a centrifugal bag on the outlet side.
(79) The device for preparing a wound healing promoter according to any one of (62) to (76), comprising extracorporeal circulation circuits on the inlet side and the outlet side of the container.
(80) The wound healing promoter preparation device according to any one of (62) to (79), which is packaged in a sterile bag and sterilized.
(81) The device for preparing a wound healing promoter according to (77), wherein the inlet / outlet of the container communicates with the outside and is packaged, and the entire apparatus is packaged in a sterilization bag and sterilized.
(82) A wound healing promoter obtained using the wound healing promoter preparation device according to any one of (62) to (81).
(83) A method for treating a wound site, wherein the wound healing promoter according to any one of (1) to (23), (61) and (82) is attached to a wound site.
(84) The method for treating a wound site according to (83), wherein the container is opened and attached in a state where one surface of the sheet-like porous body is exposed from the container.
(85) The method for treating a wound site according to (84), wherein the sheet-like porous body is taken out from the container and attached.
(86) The method for treating a wound site according to any one of (83) to (85), wherein the wound healing promoter is attached to the wound site within 30 minutes after preparation.
(87) The method for treating a wound site according to any one of (83) to (86), wherein the wound is a body surface.
(88) The method for treating a wound site according to any of (83) to (87), wherein the pasted wound healing promoter is covered and fixed with a protective material.
(89) The method for treating a wound site according to (88), wherein the protective material is a sheet made of a material having no water permeability.
(90) The method for treating a wound site according to (89), wherein the protective material is a sheet made of a material having gas permeability and no water permeability.
図１は、本発明の創傷治癒促進材による創傷部位の治療方法を示す。図１Ａは、シート状多孔質体の一面を容器から露出させた状態で貼り付ける方法を示し、図１Ｂは、容器からシート状多孔質体を取り出して貼り付ける方法を示す。 FIG. 1 shows a method for treating a wound site using the wound healing promoter of the present invention. FIG. 1A shows a method of attaching a sheet-like porous body with one surface exposed from the container, and FIG. 1B shows a method of taking out and attaching the sheet-like porous body from the container.
以下、本発明の実施の形態について詳細に説明する。
本発明で用いるシート状多孔質体とは、角形あるいはディスク状のシート材料であり、細孔あるいは繊維間隙などの多孔部を有することにより、多孔部の表面において吸着や濾過によって細胞を捕捉しうる多孔質材料である。一般に、創傷部位表面は平面と限らず凹凸をともなう場合が多いため、より高い創傷治癒効果を発現するためには、創傷部位の形状に合わせてシート状多孔質体が密着していることが効果的である。従って、本シート状多孔質体は創傷部位の形状に合わせて変形可能な柔軟性を有することが好ましく、それにより、例えば数ｃｍ四方に及ぶ大きな創傷部位であっても、一枚のシートで隙間なく被覆することができる。
また、本発明で用いるシート状多孔質体は、血液等の細胞浮遊液から少なくとも白血球および／または血小板等の血球細胞を捕捉しうる材料であるが、フィルター層としての機能を有することが好ましい。すなわち、細胞浮遊液を接触あるいは通過させた際に、少なくとも白血球および／または血小板といった血球細胞や成長因子等を捕捉し、液体部分を捕捉しないという分離機能を有していればよく、それには、サイズ分離だけではなく材料表面に対する細胞親和性（吸着性等）に基づく分離も含まれる。
シート状多孔質体は、特定の血球細胞を多く捕捉しうるという選択分離性を有するものが好ましい。この選択分離性は、多孔質材料の素材や形状を適宜選択することにより達成可能であるが、後述するように、多孔質材料の表面処理によってより高選択的に制御することもできる。本発明の創傷治癒促進材においては、例えば成長因子の産生や分泌作用に優れることから、赤血球に比して白血球および／または血小板を選択的に捕捉しうるシート状多孔質体が特に好ましい。その際、捕捉率が高いほどより少量の細胞浮遊液から目的物を得ることができるので、白血球および／または血小板の捕捉率は何れも５０％以上あれば好ましい。
シート状多孔質材料の形態としては、不織布、織布、スポンジ状構造体、粒子をシート状の袋に詰めたものなどが例示できる。この中でも、捕捉対象物として白血球や血小板を意図する場合には、除去効率という観点から、不織布やスポンジ状構造体がより好ましい。
本明細書で言う不織布とは、一層以上の繊維の塊を製編織しないで布状構造としたものを言う。繊維の素材としては、合成繊維、天然繊維、無機繊維等が用いられる。中でも疎水性高分子を主要成分とする合成繊維、例えばポリエチレンテレフタレート、ポリブチレンテレフタレート、ナイロン、ポリプロピレン、ポリエチレン、ポリスチレン、ポリアクリロニトリル等の繊維は、細胞の接着性が高いので好ましく用いられる。
シート状多孔質材料が不織布である場合の繊維の平均直径は、好ましくは０．３μｍ以上５０．０μｍ未満であり、より好ましくは０．５μｍ以上４０．０μｍ以下であり、さらに好ましくは０．７μｍ以上３５．０μｍ以下であり、さらに好ましくは１．０μｍ以上２０．０μｍ以下であり、特に好ましくは１．０μｍ以上９．０μｍ以下である。平均直径が０．３μｍより小さくなると血液細胞浮遊液や血液を不織布に流すときに流動性が悪くなって装置内の圧力損失が高くなる。また、平均直径が５０．０μｍより大きいと白血球および／または血小板の捕捉率が悪くなってしまう。また、白血球および／または血小板の捕捉率が悪くなると、結果としてシート状多孔質材料内に捕捉された細胞間の距離が大きくなってしまうため、前述のとおり産生された成長因子がより効果的に機能するためにも繊維の平均直径は上記の範囲が好ましい。
本発明で用いる不織布を構成する繊維の平均直径は、例えば不織布を構成している繊維の走査型電子顕微鏡写真を撮り、無作為に選択した１００本以上の繊維の直径を測定し、それらを数平均する方法で求められる。
本発明で用いる不織布の嵩密度は、好ましくは０．０５ｇ／ｃｍ３から０．５ｇ／ｃｍ３、更に好ましくは０．０７ｇ／ｃｍ３から０．４ｇ／ｃｍ３、より好ましくは０．１ｇ／ｃｍ３から０．３ｇ／ｃｍ３が良い。嵩密度が０．５ｇ／ｃｍ３より大きくなると細胞浮遊液や血液を不織布に流すときに流動性が悪くなって圧力損失が高くなる。また、嵩密度が０．０５ｇ／ｃｍ３より小さいと細胞捕捉率が悪くなってしまうとともに、前述のとおり捕捉された細胞間の距離が大きくなってしまうため、前述のとおり産生された成長因子がより効果的に機能するためにも繊維の嵩密度は上記の範囲が好ましい。
また本発明でいうスポンジ状構造体とは、連続開放気孔を有する三次元網目状連続組織を有する構造体をいう。スポンジ状構造体の材質は特に限定されず、セルロースやその誘導体などの天然高分子、あるいは疎水性高分子を主要成分とするポリオレフィン、ポリアミド、ポリイミド、ポリウレタン、ポリエステル、ポリサルホン、ポリアクリロニトリル、ポリエーテルサルホン、ポリ（メタ）アクリレート、ブタジエン・アクリロニトリル共重合体、エチレン・ビニルアルコール共重合体、ポリビニルアセタールもしくはそれらの混合物などの高分子材料は細胞の接着性が高いので好ましく用いられる。
スポンジ状構造体の平均孔径は好ましくは１．０μｍ以上４０μｍ以下であり、より好ましくは２．０μｍ以上３５μｍ以下であり、さらに好ましくは３．０μｍ以上３０μｍ以下である。平均直径が１．０μｍより小さくなると細胞浮遊液や血液をスポンジ状構造体に流すときに血液の流動性が悪くなり、装置内の圧力損失が高くなる。また、平均直径が４０μｍより大きいと白血球捕捉率が悪くなってしまう。また、スポンジ状構造体の平均孔径が大きくなると、結果としてフィルター層内に捕捉された細胞間の距離が大きくなってしまうため、前述のとおり産生された成長因子がより効果的に機能するためにもスポンジ状構造体の平均孔径は上記の範囲が好ましい。
本発明に言うスポンジ状構造体の平均孔径とは、水銀圧入法で測定して得られた値である。水銀圧入法（例えば島津製作所、ポアサイザ９３２０）による測定により、縦軸に細孔の容量の微分値をとり、横軸に孔径をとってグラフを描き、そのピークに当たる点（最頻値）を平均孔径とする。なお、水銀圧入法による測定値としては１〜２６５０ｐｓｉａの圧力範囲で測定した値を用いる。
本発明で用いるシート状多孔質材料の厚みは、創傷治癒効果を高めるために０．０１ｍｍから３．０ｍｍであり、好ましくは０．０５ｍｍから２．５ｍｍ、より好ましくは０．１ｍｍから２．０ｍｍである。シート状多孔質材料の厚みが３．０ｍｍより大きくなると、創傷部位への固定が困難になるとともに、白血球や血小板からの成長因子が創傷部位に届きにくくなってしまう。また、シート状多孔質材料の厚みが０．０１ｍｍより小さいと機械的強度が弱くなってしまう。細胞から産生される成長因子はその細胞自らに作用（自己分泌、ａｕｔｏｃｒｉｎｅ）し、さらに強く作用する場合と、周辺の細胞から産生され拡散した成長因子が作用（傍分泌、ｐａｒａｃｒｉｎｅ）しあうことにより、細胞間で効果が増強される場合がある。従って、成長因子をより効果的に機能させるためには細胞同士の距離がなるべく近いことが望ましいため、フィルター層の厚みは上記の範囲内であることが望ましい。また、シート状多孔質材料は一枚で上記の厚みを持つものを使用してもよいし、厚みの小さいものを２枚以上重ねて使用することもできる。
さらに、シート状多孔質材料の表面には、捕捉対象物質のみを選択的に吸着捕捉できるようにするために特定の高分子をコーティングまたはグラフト処理などをすることもできる。例えば、血球は吸着しやすいが血小板は吸着し難い公知のポリマーをコーティングまたは固定化したシート状多孔質材料と、血小板を吸着しやすいシート状多孔質材料とにこの順で血液や細胞浮遊液を処理することにより、血小板をより選択的に接着したシート状多孔質材料を調製することもできる。このようなポリマーの例としては、例えば、ＷＯ８７／０５８１２号パンフレット、ＷＯ０３／０１１９２４号パンフレットあるいはＷＯ０３／０４７６５５号パンフレット等を参照することができる。また、血球もしくは血漿タンパク質などの特定成分をより選択的に吸着しうるリガンドをコーティングまたは固定化してもよい。
シート状多孔質材料には、生分解性である素材を用いることもできる。生体吸収性の素材としては、例えばポリ乳酸、ポリグリコール酸、乳酸とグリコール酸の共重合体、ポリリンゴ酸、ポリ−ε−カプロラクトンなどのポリエステル或いはセルロース、ポリアルギン酸、キチン、キトサンなどの多糖類等を挙げることができる。これらの素材を用いることで、より生体適合性の高い創傷治癒促進材とすることができる。
本発明の創傷治癒促進材は、前記のシート状多孔質体の表面に少なくとも白血球および／または血小板が存在することが必要である。血球細胞の存在量（密度）については特に限定しないが、創傷治癒促進材としての効果をより高めるためには、一定容積の多孔質体中の血球密度が少なくとも血液中の濃度レベル以上に濃縮されていることが好ましい。また、前述の細胞間相互作用（パラクリン）の点からも、細胞密度が低すぎると成長因子産生が低下するおそれがあるので、細胞密度を高めて細胞間距離を短くすることが効果的である。これらの理由から、シート状多孔質体の表面に存在する白血球密度が６．０×１０６個／ｃｍ３以上および／または血小板密度が２．５×１０８個／ｃｍ３以上であることが好ましい。一方、血球密度が高いほど創傷治癒効果は高くなる傾向にあるが、例えば濾過操作のみによって調整する際には、目詰まりにより処理時間が増大する、あるいは多孔質体が閉塞するおそれがあるので、白血球密度の上限を６．０Ｘ１０８個／ｃｍ３以下、血小板密度の上限を１．０Ｘ１０１０個／ｃｍ３以下にすることが好ましい。
前記表面に存在する血球細胞は、末梢血、骨髄および臍帯血に由来するヒトの血球細胞であり、免疫的な拒絶や感染防除等の理由から創傷の治癒を受ける患者自身の血液に由来するもの（自己血）やヒト白血球抗原（ＨＬＡ）の型が近いもの（同種血）の血液に由来するものが好ましい。
シート状多孔質体の表面にこれらの血球細胞を捕捉させ、創傷治癒促進材を調製するための細胞浮遊液は、少なくとも白血球および／または血小板を含む浮遊液であり、末梢血、骨髄、臍帯血そのままであってもよいし、全血製剤や分離された成分製剤、あるいは予め特定の細胞分画を除去した細胞浮遊液であってもよい。これらの細胞浮遊液は、抗凝固剤として一般的なクエン酸塩、ヘパリン、低分子ヘパリン、ヘパリノイド等のヘパリン類、あるいは、フサン（ＦＵＴ）、ＦＯＹ、アルガトロバン等の水解酵素阻害剤を含んでいてもよい。また、細胞浮遊液は新鮮な状態であることが好ましく、生体から取り出したのち細胞浮遊液をシート状多孔質体によりろ過するまでの時間が４８時間以内のものがより好ましい。
シート状多孔質体の表面に存在させる血液細胞としては、幹細胞もしくは前駆細胞といった未熟な細胞ばかりでなく、成熟白血球や成熟血小板といった成熟細胞を多く含むことが望ましい。血液中や創傷組織中に少量含まれる幹細胞もしくは前駆細胞が増殖・分化して創傷組織の再生を行うさいには、単独で組織再生をするのではなく成熟細胞とのネットワークの中で創傷治癒に最適な増殖・分化を繰り返して組織再生を実現するからである。
本発明の創傷治癒促進材には線維芽細胞を組み込むこともできる。線維芽細胞は血液細胞浮遊液とは別に、シート状多孔質材料と接触させることで組み込むこともできるが、予め血液細胞を含む細胞浮遊液に混合させてから、シート状多孔質材料と接触させてもよい。また、線維芽細胞のみをさらに別の材料（フィルター層）に付着させた後、血液細胞を付着させたシート状多孔質材料と組み合わせることもできる。線維芽細胞はとくに創傷部位に含まれるものと同じ線維芽細胞を組み込むことが好ましく、これにより創傷部位の治癒を促進できる。
本発明の創傷治癒促進材は、その調製過程で培養することもできる。培養液は特に限定されず、通常細胞培養で用いられるものであればよい。さらに血液細胞を活性化する因子を加えて培養することもできる。例えば、トロンビン等の血小板活性化剤を入れてから培養することで局所的な成長因子の濃度を高めることができる。
本発明の創傷治癒促進材には、さらにフィブリンを含んでいてもよい。フィブリンを調製するためのフィブリノーゲン溶液としては特に特定されないが、製剤として市販されているものを使用することができる。また血液細胞浮遊液として血液を用いた際にシート状多孔質材料からドレインとして回収される液を遠心し、これを濃縮して用いることもできる。また、線維芽細胞を含む場合には、細胞は予めフィブリンに包埋する形態でもよいし、フィブリンに後から細胞のみを播種してもよい。フィブリンを含むことで、創傷部位への創傷治癒促進材の固定化が容易となるうえに、フィブリンゲル内に含まれる他の成長因子によりさらに創傷の治癒促進効果を上げることができる。
本発明の創傷治癒促進材を調製するには、細胞浮遊液に含まれる少なくとも白血球および／または血小板を前記シート状多孔質体の表面に捕捉させることが必要である。この捕捉工程においては、例えば、シート状多孔質体を広げて細胞浮遊液を直接滴下し濾過する、ラボ装置で用いられる適当な濾過ホルダーに把持して細胞浮遊液を濾過する（例えば図１Ｂを参照できる）、あるいはシステムの説明で後述する開放可能な液密容器にセットして濾過すればよい（例えば図１Ａおよび図１Ｂを参照できる）。後者二つの場合は、自由落下でもよく、ポンプやシリンジ等を用いて強制的に濾過してもよい。また、単に濾過するだけではなく、細胞浮遊液が接触した状態で一定時間保持する等の操作を併用すれば、血球細胞の表面捕捉率を高めることもできる。
濾過操作においては、特に断続的な濾過操作を何度も行うと濾過のシェアストレスで細胞が活性化してしまい、ドレインの血漿中に成長因子を放出してしまうことが懸念される。従って、細胞浮遊液をワンスルーで濾過することが好ましく、準備や操作が簡便でしかも処理時間が短いという利点も得られる。また、細胞浮遊液を濾過する方向は一方向であることが好ましい。例えば、何らかの原因により、シート状多孔質体中に十分な密度の白血球および／または血小板を捕捉できない場合には、シート状多孔質体の両面で細胞密度が異なり、濾過入口側の細胞密度が高くなることがある。そのような場合には、濾過入口側の面を創傷部位に密着することで捕捉細胞の創傷治癒促進効果をより効率的に発現させることができるからである。あるいは、意図的に密度を偏在させてこのような作用を狙うこともできる。
このようなワンスルー濾過を行う場合は、輸血における白血球除去操作のように大量の細胞浮遊液（一単位である４００ｍｌ以上）を処理することも可能ではあるが、少量で濾過を行うほど処理時間が短縮されるので、例えば創傷の処置に平行して準備が必要な場合には極めて有用となる。従って、細胞浮遊液の濾過液量を４００ｍｌ未満とすることが好ましく、また、シート状多孔質体の有効濾過面積に対する濾過液量の比を１０ｍｌ／ｃｍ２未満とする、あるいはシート状多孔質体の容積に対する濾過液量の比を１００未満としても濾過時間が短くなるので好ましい。
一方、開放可能な液密容器を用いれば、体外循環により濾過することも容易である。この場合は、処理時間は比較的長くなるものの、大量の血液を連続的に循環し濾過し続けることにより、シート状多孔質体における血球密度が高くなるという利点がある。また、システムの説明で後述するように、シート状多孔質体を反物上に巻いて円筒状の容器に組み込むと、１〜２ｍ２の濾過面積を容易に確保できるため、大量の血液からきわめて大面積の創傷治癒促進材を得ることができる。その際、体外循環における濾過流速は２０〜２００ｍｌ／ｍｉｎ、濾過時間は１０〜３００分であれば好ましい。体外循環により濾過を行う場合には濾過流速は２０〜２００ｍＬ／分が好ましい。より好ましくは２５〜１５０ｍＬ／分、さらに好ましくは３０〜１２０ｍＬ／分である。濾過流速がこれより低いと抗凝固剤の種類によっては体外循環施行中に血液が凝固しやすくなるし、体外循環時間が長くなって患者に負担がかかってしまう。また、濾過流速がこれより高いと、患者の血管状態によっては血液採取が困難になることがあり、さらにシート状多孔質体にかかる負荷が高くなって血液凝固を促進してしまうことが起こりうる。また、体外循環の濾過時間としては１０分〜３００分が好ましい。より好ましくは２０分〜２５０分、さらに好ましくは３０分〜２００分である。体外循環の濾過時間がこれより短くなると十分な細胞が捕捉できないおそれがあり、反対にこれより体外循環の濾過時間が長くなると患者に与える負担が大きくなる。
以上の工程により、シート状多孔質体の表面に少なくとも白血球および／または血小板が存在する本発明の創傷治癒促進材が得られるが、この調製調整方法においては、さらに洗浄工程を含んでもよい。洗浄は、例えばリン酸緩衝液や生理食塩水等の生理的水溶液を、細胞浮遊液の濾過操作と同様に実施すればよい。洗浄することにより、創傷治癒に不要な細胞や残留血液等を除去することができる。洗浄後の創傷治癒促進材については、ホルダーや容器に取り付けた状態または取り出した状態で、乾燥しないように且つ無菌的に保存すればよく、その方法は何ら限定しない。
本発明はさらに、本発明の創傷治癒促進材を調製するためのデバイスであって、液体の注入と排出のための入口と出口が付いた開放可能な液密容器の内部に、容器内部を二室に隔絶し、一方を入口側に他方を出口側に通じるようにシート状多孔質体を配置した創傷治癒促進材の調製デバイスにも関する。
本発明において、液体の注入と排出のための入口と出口が付いた液密容器とは、細胞浮遊液の注入と排出（濾過）または体外循環が可能な入口と出口が付いた容器である。例えば、公知の平板状の白血球除去フィルターデバイス（例えばＷＯ０１／０９１８８０号パンフレットを参照できる）や公知の円筒状の白血球除去モジュール（例えばＷＯ９９／０５８１７２号パンフレットを参照できる）のような外部形状を有しており、内部構造についてもこれらと同様に、濾材（本発明でいうシート状多孔質体）が容器内部を濾過前後の液体が混合しないように二室に隔絶し、一方が入口側に、他方が出口側に通じるように配置されている。
本発明の装置に使用される容器の形状は特に限定されず、シート状多孔質材料を収納しやすく、液密になっており、細胞浮遊液がシート状多孔質材料を流れやすい形状であればよい。但し、シート状の多孔質材料をそのまま収容するのであれば平板型が好ましく、反物状にして収容するのであれば円筒型が収納しやすい。さらに、平板型容器に可撓性材料を使用し、平板型容器全体を反物状に巻くことによりコンパクトな状態とすることもできる。
ここでいう開放可能とは、血球が捕捉されたシート状多孔質材料を前記容器から簡便に取り出し可能、あるいは血球捕捉面の一部を容易に露出可能な容器構造を意味し、本デバイスにおいて必要な構造である。例えば、容器本体がネジ式、パッキング式、すり合わせ式等の締結手段を有するもの、あるいは引き剥がし可能な貼り合せ式になっている構造を例示できる。より具体的には、平板状容器の場合は、液体入口が付いた可撓性樹脂シート容器と液体出口が付いた可撓性樹脂シート容器とを、シート状多孔質体を挟み込むようにそれらの周縁部で溶着または接着した軟質デバイスが好ましく、円筒状容器の場合は、液体入口と前記の締結手段が付いたヘッダー部と、液体出口と締結手段がついたヘッダー部とを、反物状にしたシート状多孔質体を収容した筒状胴部の両端に締結した硬質デバイスが好ましい。前者においては、例えば図１Ａに示すように、溶着または接着したシート血球が捕捉されたシート状多孔質体の一部を露出して創傷部位に貼り付ける際に非常に便利であり、後者においては、全体としては小型でも大きな濾過面積を確保できるので体外循環により調製するのに適しており、その結果、大面積あるいは大量の創傷治癒促進材を得るのに適している。
なお、血球が捕捉されたシート状多孔質材料を前記容器から簡便に取り出す、あるいは血球捕捉面の一部を容易に露出可能であれば、前記のような開放可能な手段がなくてもよく、容器の全体もしくは一部が外側から容易に破壊可能な材料や構造からなっていてもよい。さらには、市販されている平板状または円筒状の白血球除去フィルターを適宜選択して利用することもできる。
本発明の創傷治癒促進材調製デバイスには、細胞浮遊液を無菌的に流すために容器入口側に採血バッグと接続するためのスパイク針付きのチューブを接続することもできる。単にチューブだけを取り付けておき、無菌接続器で接続してもよい。また、ドレイン液を用いて血漿又は血清を調製することを容易にするために容器の出口側に遠心用のバッグを接続することもできる。さらに、血液ポンプや導血部および返血部等を備えた一般的な体外循環回路を接続することもできる。
本発明の創傷治癒促進材調製デバイスを滅菌袋に包装する場合、シート状多孔性材料を収容する容器を滅菌袋に包装し、さらに装置全体を別の滅菌袋で包装してもよい。こうすることにより、細胞浮遊液を流した後に多孔質材料を実際に創傷部位に貼り付けるまでの間容器および多孔質材料を無菌的に保持可能となり、取扱い性が著しく向上する。
本発明はさらに、本発明の創傷治癒促進材を創傷部位に貼り付ける創傷部位の治療方法にも関する。
本発明の創傷治癒促進材による創傷部位の治療方法には、容器からシート状多孔質体を取り出して貼り付ける方法（図１Ｂ）や、シート状多孔質体の一面を容器から露出させた状態で貼り付ける方法（図１Ａ）が例示できる。前者においては、露出面の反対側が容器で覆われているのでシート状多孔質体の無菌性により優れており、また多孔質体の乾燥を防止することもできる。このように理由から、前者においては特に体表面への適用性に優れている。
また、本発明の治療方法においては、創傷部位に貼り付けた創傷治癒促進材の表面を保護材により被覆固定してもよい。その際、治療部位、特に創傷治癒促進材表面の乾燥を防ぐために、透水性を有さない素材からなる保護材を用いることが好ましい。また、ガス透過性を有しかつ透湿性を有さない素材であれば、血球細胞への酸素供給が容易になり、細胞活性がより長期間維持されるので特に好ましい。これらの保護材は、創傷治療治癒において通常用いられるものを利用すればよい。
以下の実施例により本発明をより具体的に説明するが、本発明は実施例によって限定されるものではない。Hereinafter, embodiments of the present invention will be described in detail.
The sheet-like porous material used in the present invention is a square or disk-like sheet material, and has a porous part such as a pore or a fiber gap, so that cells can be captured by adsorption or filtration on the surface of the porous part. It is a porous material. In general, the surface of the wound site is not limited to a flat surface and often has irregularities, so in order to express a higher wound healing effect, it is effective that the sheet-like porous body closely adheres to the shape of the wound site Is. Accordingly, it is preferable that the sheet-like porous body has a flexibility that can be deformed according to the shape of the wound site. It can coat without.
The sheet-like porous material used in the present invention is a material that can capture at least blood cells such as white blood cells and / or platelets from a cell suspension such as blood, but preferably has a function as a filter layer. That is, when the cell suspension is contacted or passed, it is sufficient to have a separation function of capturing at least blood cells such as leukocytes and / or platelets and growth factors, and not capturing the liquid part. In addition to size separation, separation based on cell affinity (adsorbability, etc.) to the material surface is also included.
The sheet-like porous body preferably has a selective separation property capable of capturing many specific blood cells. This selective separability can be achieved by appropriately selecting the material and shape of the porous material, but can be controlled more selectively by surface treatment of the porous material, as will be described later. In the wound healing promoter of the present invention, for example, a sheet-like porous body capable of selectively capturing leukocytes and / or platelets as compared with erythrocytes is particularly preferable because of excellent production and secretion of growth factors. At that time, since the target can be obtained from a smaller amount of cell suspension as the capture rate is higher, it is preferable that the capture rate of leukocytes and / or platelets is 50% or more.
Examples of the form of the sheet-like porous material include nonwoven fabrics, woven fabrics, sponge-like structures, and those in which particles are packed in a sheet-like bag. Among these, when white blood cells or platelets are intended as the capture target, a nonwoven fabric or a sponge-like structure is more preferable from the viewpoint of removal efficiency.
The non-woven fabric referred to in the present specification refers to a non-woven fabric having a cloth-like structure without being knitted or woven. Synthetic fibers, natural fibers, inorganic fibers, etc. are used as the fiber material. Among them, synthetic fibers containing a hydrophobic polymer as a main component, for example, fibers such as polyethylene terephthalate, polybutylene terephthalate, nylon, polypropylene, polyethylene, polystyrene, and polyacrylonitrile are preferably used because of their high cell adhesion.
When the sheet-like porous material is a nonwoven fabric, the average diameter of the fibers is preferably 0.3 μm or more and less than 50.0 μm, more preferably 0.5 μm or more and 40.0 μm or less, and even more preferably 0.7 μm. It is 35.0 μm or less, more preferably 1.0 μm or more and 20.0 μm or less, and particularly preferably 1.0 μm or more and 9.0 μm or less. When the average diameter is smaller than 0.3 μm, the fluidity is deteriorated when the blood cell suspension or blood is passed through the nonwoven fabric, and the pressure loss in the apparatus is increased. On the other hand, when the average diameter is larger than 50.0 μm, the capture rate of leukocytes and / or platelets is deteriorated. In addition, if the capture rate of leukocytes and / or platelets deteriorates, the distance between the cells trapped in the sheet-like porous material increases as a result, so that the growth factor produced as described above is more effective. In order to function, the average diameter of the fibers is preferably within the above range.
The average diameter of the fibers constituting the nonwoven fabric used in the present invention is measured, for example, by taking a scanning electron micrograph of the fibers constituting the nonwoven fabric, measuring the diameters of 100 or more randomly selected fibers, and counting them. Calculated by averaging method.
The bulk density of the nonwoven fabric used in the present invention is preferably 0.05 g / cm 3 from 0.5 g / cm 3, more preferably 0.07 g / cm 3 from 0.4 g / cm 3, more preferably 0.1 g / cm 3 to 0.3 g / cm 3 is preferable. When the bulk density is higher than 0.5 g / cm 3 , the fluidity is deteriorated when the cell suspension or blood is passed through the nonwoven fabric, and the pressure loss is increased. In addition, when the bulk density is less than 0.05 g / cm 3 , the cell capture rate is deteriorated and the distance between the captured cells is increased as described above. In order to function more effectively, the bulk density of the fibers is preferably in the above range.
The sponge-like structure referred to in the present invention refers to a structure having a three-dimensional network-like continuous structure having continuous open pores. The material of the sponge-like structure is not particularly limited, and is a polyolefin, polyamide, polyimide, polyurethane, polyester, polysulfone, polyacrylonitrile, polyethersulfur, which is mainly composed of natural polymers such as cellulose and its derivatives, or hydrophobic polymers. Polymer materials such as phon, poly (meth) acrylate, butadiene / acrylonitrile copolymer, ethylene / vinyl alcohol copolymer, polyvinyl acetal or a mixture thereof are preferably used because of their high cell adhesion.
The average pore diameter of the sponge-like structure is preferably 1.0 μm or more and 40 μm or less, more preferably 2.0 μm or more and 35 μm or less, and further preferably 3.0 μm or more and 30 μm or less. When the average diameter is smaller than 1.0 μm, the fluidity of blood deteriorates when the cell suspension or blood flows through the sponge-like structure, and the pressure loss in the apparatus increases. On the other hand, when the average diameter is larger than 40 μm, the leukocyte capturing rate is deteriorated. In addition, when the average pore size of the sponge-like structure is increased, the distance between the cells trapped in the filter layer is increased as a result, so that the growth factor produced as described above functions more effectively. The average pore diameter of the sponge-like structure is preferably in the above range.
The average pore diameter of the sponge-like structure referred to in the present invention is a value obtained by measurement by a mercury intrusion method. By measuring with the mercury intrusion method (for example, Shimadzu Corp., Pore Sizer 9320), the vertical axis represents the pore volume differential value, the horizontal axis represents the pore diameter, the graph is drawn, and the point corresponding to the peak (mode) is averaged. The hole diameter. In addition, as a measured value by the mercury intrusion method, a value measured in a pressure range of 1 to 2650 psia is used.
The thickness of the sheet-like porous material used in the present invention is 0.01 mm to 3.0 mm, preferably 0.05 mm to 2.5 mm, more preferably 0.1 mm to 2.0 mm in order to enhance the wound healing effect. It is. When the thickness of the sheet-like porous material is larger than 3.0 mm, fixing to the wound site becomes difficult, and growth factors from white blood cells and platelets are difficult to reach the wound site. Further, if the thickness of the sheet-like porous material is smaller than 0.01 mm, the mechanical strength becomes weak. A growth factor produced from a cell acts on the cell itself (autocrine, autocrine), and when it acts more strongly, and a growth factor produced from a surrounding cell diffuses and acts (paracrine). In some cases, the effect is enhanced between cells. Therefore, in order to make the growth factor function more effectively, it is desirable that the distance between the cells is as close as possible. Therefore, the thickness of the filter layer is preferably within the above range. In addition, the sheet-like porous material may be a single sheet having the above thickness, or two or more sheets having a small thickness may be used in an overlapping manner.
Furthermore, the surface of the sheet-like porous material can be coated or grafted with a specific polymer so that only the substance to be captured can be selectively adsorbed and captured. For example, blood or cell suspension is applied in this order to a sheet-like porous material that is coated or immobilized with a known polymer that easily adsorbs blood cells but not platelets, and a sheet-like porous material that easily adsorbs platelets. By processing, a sheet-like porous material to which platelets are more selectively adhered can also be prepared. Examples of such polymers can be referred to, for example, WO87 / 05812 pamphlet, WO03 / 011924 pamphlet, WO03 / 047655 pamphlet and the like. In addition, a ligand capable of more selectively adsorbing specific components such as blood cells or plasma proteins may be coated or immobilized.
For the sheet-like porous material, a biodegradable material can also be used. Examples of bioabsorbable materials include polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, polyesters such as polymalic acid and poly-ε-caprolactone, or polysaccharides such as cellulose, polyalginic acid, chitin, and chitosan. Can be mentioned. By using these materials, a wound healing promoter with higher biocompatibility can be obtained.
The wound healing promoter of the present invention requires that at least leukocytes and / or platelets are present on the surface of the sheet-like porous body. The abundance (density) of blood cells is not particularly limited, but in order to further enhance the effect as a wound healing promoter, the blood cell density in a certain volume of porous material is concentrated at least above the concentration level in blood. It is preferable. Also, from the viewpoint of the above-mentioned cell-cell interaction (paracrine), if the cell density is too low, growth factor production may be reduced. Therefore, it is effective to increase the cell density and shorten the intercellular distance. . For these reasons, the density of leukocytes existing on the surface of the sheet-like porous body is 6.0 × 10 6 cells / cm 3 or more and / or the platelet density is 2.5 × 10 8 cells / cm 3 or more. preferable. On the other hand, as the blood cell density is higher, the wound healing effect tends to be higher, but for example, when adjusting only by filtration operation, the treatment time may increase due to clogging, or the porous body may be clogged, It is preferable that the upper limit of the white blood cell density is 6.0 × 10 8 cells / cm 3 or less and the upper limit of the platelet density is 1.0 × 10 10 cells / cm 3 or less.
The blood cells present on the surface are human blood cells derived from peripheral blood, bone marrow and umbilical cord blood, and are derived from the patient's own blood undergoing wound healing for reasons such as immune rejection and infection control Those derived from blood of (autologous blood) and human leukocyte antigen (HLA) of similar types (allogeneic blood) are preferred.
A cell suspension for capturing these blood cells on the surface of the sheet-like porous body and preparing a wound healing promoter is a suspension containing at least leukocytes and / or platelets. Peripheral blood, bone marrow, umbilical cord blood It may be used as it is, or may be a whole blood preparation, a separated component preparation, or a cell suspension from which a specific cell fraction has been removed in advance. These cell suspensions contain common citrates, heparins, low molecular weight heparins, heparinoids and other heparins as anticoagulants, or hydrolytic enzyme inhibitors such as fusan (FUT), FOY, and argatroban. Also good. In addition, the cell suspension is preferably in a fresh state, and it is more preferable that the time until the cell suspension is filtered through the sheet-like porous body after being taken out from the living body is within 48 hours.
It is desirable that the blood cells present on the surface of the sheet-like porous body contain not only immature cells such as stem cells or progenitor cells but also many mature cells such as mature leukocytes and mature platelets. When stem cells or progenitor cells contained in small amounts in the blood or wound tissue proliferate and differentiate to regenerate the wound tissue, it is not necessary to regenerate the tissue alone but to repair the wound in a network with mature cells. This is because tissue regeneration is realized by repeating optimal proliferation and differentiation.
Fibroblasts can also be incorporated into the wound healing promoter of the present invention. Fibroblasts can be incorporated by contacting with a sheet-like porous material separately from the blood cell suspension, but after mixing with a cell suspension containing blood cells in advance, the fibroblasts are brought into contact with the sheet-like porous material. May be. Alternatively, only fibroblasts may be attached to another material (filter layer) and then combined with a sheet-like porous material to which blood cells are attached. The fibroblasts preferably incorporate the same fibroblasts as those contained in the wound site, which can promote healing of the wound site.
The wound healing promoter of the present invention can also be cultured during the preparation process. The culture solution is not particularly limited as long as it is usually used in cell culture. Furthermore, it can also culture | cultivate by adding the factor which activates a blood cell. For example, local growth factor concentrations can be increased by culturing after adding a platelet activating agent such as thrombin.
The wound healing promoter of the present invention may further contain fibrin. Although it does not specifically specify as a fibrinogen solution for preparing fibrin, what is marketed as a formulation can be used. In addition, when blood is used as the blood cell suspension, the liquid recovered as a drain from the sheet-like porous material can be centrifuged and used after being concentrated. When fibroblasts are included, the cells may be pre-embedded in fibrin, or only cells may be seeded later on fibrin. By including fibrin, the wound healing promoter can be easily fixed to the wound site, and the wound healing promoting effect can be further enhanced by other growth factors contained in the fibrin gel.
In order to prepare the wound healing promoter of the present invention, it is necessary to capture at least leukocytes and / or platelets contained in the cell suspension on the surface of the porous sheet. In this capturing step, for example, the cell-like suspension is spread and the cell suspension is directly dropped and filtered. The cell suspension is filtered by holding it in a suitable filtration holder used in a laboratory apparatus (for example, see FIG. 1B). Or may be set in an openable liquid-tight container described later in the description of the system and filtered (for example, see FIGS. 1A and 1B). In the latter two cases, free fall may be used, or filtration may be forcibly performed using a pump, a syringe, or the like. Moreover, the surface capture rate of blood cells can be increased by using not only filtration but also operation such as holding for a certain period of time in contact with the cell suspension.
In the filtration operation, especially when intermittent filtration operation is performed many times, the cell is activated by the shear stress of filtration, and there is a concern that the growth factor is released into the plasma of the drain. Therefore, it is preferable to filter the cell suspension one-through, and there is an advantage that the preparation and operation are simple and the processing time is short. Moreover, it is preferable that the direction which filters a cell suspension is one direction. For example, if for some reason it is not possible to capture sufficiently dense white blood cells and / or platelets in the sheet porous body, the cell density is different on both sides of the sheet porous body, and the cell density on the filtration inlet side is high. May be. In such a case, the wound healing promoting effect of the trapped cells can be expressed more efficiently by bringing the surface on the filtration inlet side into close contact with the wound site. Alternatively, the density can be intentionally unevenly distributed to aim at such an action.
When performing such one-through filtration, it is possible to process a large amount of cell suspension (400 ml or more as a unit) as in the leukocyte removal operation in blood transfusion, but the processing time increases as the filtration is performed in a small amount. Because it is shortened, it is extremely useful, for example, when preparation is required in parallel with wound treatment. Accordingly, it is preferable that the amount of filtrate of the cell suspension is less than 400 ml, and the ratio of the amount of filtrate to the effective filtration area of the sheet-like porous body is less than 10 ml / cm 2 or the sheet-like porous body. Even if the ratio of the amount of the filtrate to the volume of less than 100 is preferable, the filtration time is shortened.
On the other hand, if an openable liquid-tight container is used, it is easy to filter by extracorporeal circulation. In this case, although the treatment time is relatively long, there is an advantage that the blood cell density in the sheet-like porous body is increased by continuously circulating and filtering a large amount of blood. Further, as will be described later in the description of the system, when a sheet-like porous body is wound on a fabric and incorporated in a cylindrical container, a filtration area of 1 to 2 m 2 can be easily secured, so that a very large amount of blood can be obtained. An area-wide wound healing promoter can be obtained. In that case, the filtration flow rate in extracorporeal circulation is preferably 20 to 200 ml / min, and the filtration time is preferably 10 to 300 minutes. When filtration is performed by extracorporeal circulation, the filtration flow rate is preferably 20 to 200 mL / min. More preferably, it is 25-150 mL / min, More preferably, it is 30-120 mL / min. If the filtration flow rate is lower than this, depending on the type of the anticoagulant, blood tends to coagulate during the extracorporeal circulation, and the extracorporeal circulation time becomes longer and burdens the patient. Also, if the filtration flow rate is higher than this, it may be difficult to collect blood depending on the blood vessel state of the patient, and further, the load on the sheet-like porous body may increase and promote blood coagulation. . The filtration time for extracorporeal circulation is preferably 10 minutes to 300 minutes. More preferably, it is 20 minutes-250 minutes, More preferably, it is 30 minutes-200 minutes. If the filtration time for extracorporeal circulation is shorter than this, sufficient cells may not be captured, and conversely, if the filtration time for extracorporeal circulation is longer than this, the burden on the patient increases.
By the above steps, the wound healing promoter of the present invention in which at least leukocytes and / or platelets are present on the surface of the sheet-like porous body is obtained, but this preparation adjustment method may further include a washing step. The washing may be performed, for example, using a physiological aqueous solution such as a phosphate buffer or physiological saline in the same manner as the filtration operation of the cell suspension. By washing, cells unnecessary for wound healing, residual blood, and the like can be removed. The wound healing promoter after washing may be stored aseptically so as not to be dried in a state of being attached to or removed from a holder or container, and the method is not limited at all.
The present invention further provides a device for preparing the wound healing promoter of the present invention, wherein the container interior is disposed in an openable liquid-tight container having an inlet and an outlet for injecting and discharging liquid. The present invention also relates to a device for preparing a wound healing promoter, in which a sheet-like porous body is disposed so as to be isolated from a chamber and lead one to the inlet side and the other to the outlet side.
In the present invention, a liquid-tight container having an inlet and an outlet for injecting and discharging a liquid is a container having an inlet and an outlet capable of injecting and discharging (filtering) or extracorporeal circulation of a cell suspension. For example, it has an external shape such as a known flat plate-shaped leukocyte removal filter device (for example, refer to WO01 / 091880 pamphlet) or a known cylindrical leukocyte removal module (for example, refer to WO99 / 058172 pamphlet). In the same way as for the internal structure, the filter medium (sheet-like porous body in the present invention) is separated into two chambers so that the liquid before and after filtration is not mixed inside the container, one on the inlet side and the other Is arranged to communicate with the outlet side.
The shape of the container used in the apparatus of the present invention is not particularly limited, as long as it is easy to store the sheet-like porous material and is liquid-tight, and the cell suspension can easily flow through the sheet-like porous material. Good. However, a flat plate type is preferable if the sheet-like porous material is stored as it is, and a cylindrical type is easy to store if the sheet-like porous material is stored in the shape of a cloth. Furthermore, a flexible material can be used for the flat plate container, and the entire flat plate container can be wound into a reciprocal shape to achieve a compact state.
The term “openable” as used herein means a container structure in which a sheet-like porous material in which blood cells have been captured can be easily taken out from the container, or a part of the blood cell capturing surface can be easily exposed. Structure. For example, the container main body can be exemplified by a screw type, a packing type, a type having a fastening means such as a laminating type, or a structure in which the container body is a peelable type. More specifically, in the case of a flat container, a flexible resin sheet container with a liquid inlet and a flexible resin sheet container with a liquid outlet are arranged so that the sheet-like porous body is sandwiched between them. A soft device welded or bonded at the periphery is preferable. In the case of a cylindrical container, the header portion with the liquid inlet and the fastening means and the header portion with the liquid outlet and the fastening means are made into a parabolic shape. A rigid device fastened to both ends of the cylindrical body portion containing the sheet-like porous body is preferable. In the former, for example, as shown in FIG. 1A, it is very convenient to expose a part of the sheet-like porous body in which the welded or adhered sheet blood cells are captured and apply it to the wound site. As a whole, a large filtration area can be secured even with a small size, so that it is suitable for preparation by extracorporeal circulation. As a result, it is suitable for obtaining a large area or a large amount of wound healing promoter.
In addition, if the sheet-like porous material in which blood cells are captured can be easily taken out from the container, or if a part of the blood cell capturing surface can be easily exposed, there is no need for such an openable means, The whole or part of the container may be made of a material or structure that can be easily broken from the outside. Furthermore, a commercially available flat plate or cylindrical leukocyte removal filter can be appropriately selected and used.
To the wound healing promoter preparation device of the present invention, a tube with a spike needle for connecting to a blood collection bag can be connected to the container inlet side in order to aseptically flow the cell suspension. Only a tube may be attached and connected with a sterile connector. In addition, a centrifuge bag can be connected to the outlet side of the container to facilitate the preparation of plasma or serum using the drain solution. Furthermore, a general extracorporeal circuit including a blood pump, a blood introduction unit, a blood return unit, and the like can be connected.
When the wound healing promoter preparation device of the present invention is packaged in a sterile bag, the container containing the sheet-like porous material may be packaged in a sterile bag, and the entire apparatus may be packaged in another sterile bag. By doing so, the container and the porous material can be aseptically held until the porous material is actually attached to the wound site after flowing the cell suspension, and the handleability is remarkably improved.
The present invention further relates to a method for treating a wound site in which the wound healing promoter of the present invention is applied to a wound site.
In the method for treating a wound site using the wound healing promoter of the present invention, the sheet-like porous body is taken out and pasted from the container (FIG. 1B), or one surface of the sheet-like porous body is exposed from the container. A method of pasting (FIG. 1A) can be exemplified. In the former, since the opposite side of the exposed surface is covered with a container, the sterility of the sheet-like porous body is superior, and drying of the porous body can also be prevented. For this reason, the former is particularly excellent in applicability to the body surface.
Further, in the treatment method of the present invention, the surface of the wound healing promoting material affixed to the wound site may be covered and fixed with a protective material. At that time, it is preferable to use a protective material made of a material having no water permeability in order to prevent drying of the treatment site, particularly the surface of the wound healing promoter. In addition, a material that has gas permeability and does not have moisture permeability is particularly preferable because oxygen supply to blood cells is facilitated and cell activity is maintained for a longer period. What is necessary is just to utilize what is normally used in wound treatment healing as these protective materials.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
〔実施例１〕：創傷治癒促進材の調製（ヒト血液）
血液細胞を表面に有するシート状多孔質体を以下の方法で調製した。 健常人のボランティアより、抗凝固剤としてＣＰＤ（ｃｉｔｒａｔｅ−ｐｈｏｓｐｈａｔｅ−ｄｅｘｔｒｏｓｅ）を用いて末梢血を採取した（血液：ＣＰＤ＝４００：５６、白血球数：５，１００個／μＬ、血小板数：１４．４ｘ１０４個／μＬ）。ポリエチレンテレフタレート製の不織布（旭化成株式会社、平均繊維径２．６μｍ、厚み０．３８ｍｍ、嵩密度０．２７ｇ／ｃｍ３）を２５ｍｍ径に打ちぬき、これを２枚重ねてカラム（アドバンテック社、型番ＰＰ−２５）にて保持し、カラムに末梢血５ｍＬを流した。血液を流した後に連続して、カラムにリン酸緩衝液１０ｍＬを流して洗浄し、血液細胞を表面に付着させたシート状多孔質体を含む創傷治癒促進材を得た。
上記操作を別々に２回施行したが、処理に要する時間はいずれも１０分以内であった。
処理前後の血液中に含まれる白血球、血小板数を血球計算盤により測定した。
不織布フィルター層による白血球および血小板の捕捉率は以下の表１の通りであった。
血液細胞を表面に有するシート状多孔質体を以下の方法で調製した。健常人のボランティアより、抗凝固剤としてヘパリンを用いて末梢血を採取した（血液：ヘパリン＝１００：１、白血球数：３，８００個／μＬ、血小板数：２２．５ｘ１０４個／μＬ）。ポリエチレンテレフタレート製の不織布（旭化成株式会社、平均繊維径１．１μｍ、厚み０．２４ｍｍ、嵩密度０．１７ｇ／ｃｍ３）を２５ｍｍ径に打ちぬき、これを２枚重ねてカラム（アドバンテック社、型番ＰＰ−２５）にて保持し、カラムに末梢血５ｍＬを流した。 血液を流した後に連続して、カラムにリン酸緩衝液１０ｍＬを流して洗浄し、血液細胞を表面に付着させたシート状多孔質体から成る創傷治癒促進材を得た。
上記操作を別々に２回施行したが、処理に要する時間はいずれも１０分以内であった。
処理前後の血液中に含まれる白血球、血小板数を血球計算盤により測定した。
不織布フィルター層による白血球および血小板の捕捉率は以下の表２の通りであった。
血液細胞を表面に有するシート状多孔質体を以下の方法で調製した。
２型糖尿病モデルマウス（Ｃ５７ＢＬ／ＫｓＪ−ｄｂ／ｄｂ Ｊｃｔ，日本クレア（株））より、抗凝固剤としてヘパリンを用いて末梢血を採取した（白血球数：５，２００個／μＬ、血液：ヘパリン＝１００：１）。ポリ乳酸製の不織布（旭化成株式会社、平均繊維径１．１４μｍ、厚み０．２０ｍｍ、嵩密度０．２０ｇ／ｃｍ３）を１３ｍｍ径に打ちぬき、これを３枚重ねて５ｍＬシリンジ（テルモ社）にて保持し、これにマウス末梢血２００μＬを流した。 血液を流した後に連続して、リン酸緩衝液２００μＬを流して洗浄し、血液細胞を表面に付着させたシート状多孔質体から成る創傷治癒促進材を得た。
上記操作を別々に２回施行したが、処理に要する時間はいずれも２分以内であった。
処理前後の血液中に含まれる白血球を血球計算盤により測定した。
不織布フィルター層による白血球の捕捉率は以下の表３の通りであった。
実施例１にて調製した血液細胞を表面に付着した不織布創傷治癒促進材と、線維芽細胞とを共培養して、線維芽細胞の増殖率を検討した。
培養容器としては、３μｍのポアサイズの膜で隔てられたインサートを有するマルチプルウェル培養用プレート（コーニング社、トランスウェル３４５２）を用いた。
インサートの膜で隔てられた上側に実施例１にて血液処理を行った不織布（即ち、血液細胞を表面に付着した本発明の創傷治癒促進材）２枚を入れ、下側にヒト胎児肺由来の正常二倍体線維芽細胞株（ＨＥＬ、ｈｕｍａｎ ｅｍｂｒｙｏｎｉｃ ｌｕｎｇ ｆｉｂｒｏｂｌａｓｔ）を播種した。細胞の播種密度は、各１ｘ１０５個／ウエルとした。
Ｂａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ（Ｓｉｇｍａ社、Ｂ１５２２）にメーカー指定の通り、ピルビン酸ナトリウム（ＩＣＮ社）を１ｍＭ、１００倍濃縮非必須アミノ酸（ＩＣＮ社、Ｃａｔ．＃１６８１０４９）を１００倍希釈となるように、そしてグルタミンを２ｍＭとなるように添加した。これにさらに、最終濃度が１０％となるように牛胎児血清（ＦＣＳ、ＳｔｅｍＣｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ社）と、抗生物質としてペニシリン及びストレプトマイシンを添加して、培養液を作製した。培養液の容量は４ｍＬ／ウエルとした。
また、コントロールとしては、血液で処理していない不織布とＨＥＬとの共培養を行った。
上記の培養物を、５％ ＣＯ２、３７℃のインキュベーターで４日間培養後、線維芽細胞の数を測定した。結果を表４に示す。線維芽細胞の回収には０．２５％ Ｔｒｙｐｓｉｎ／ＥＤＴＡ液（ギブコ社）を用いた。
〔試験例２〕：創傷治癒促進材からの成長因子産生
実施例２にて調製した血液細胞を表面にした不織布創傷治癒促進材を培養液にて培養し付着した細胞から産生される各種増殖因子の濃度を検討した。
培養容器としては、直径３５ｍｍの培養ディッシュ（旭テクノグラス社）を用いた。
実施例２にて血液処理を行った不織布（即ち、血液細胞を表面に付着した本発明の創傷治癒促進材）２枚を入れた。
市販の培養液Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（Ｄ−ＭＥＭ、ギブコ社）に最終濃度が２％となるように牛胎児血清（ＦＢＳ、ギブコ社）と、抗生物質としてゲンタマイシンを添加して、培養液を作製した。培養液の容量は２ｍＬとした。
上記の培養物を、５％ＣＯ２、３７℃のインキュベーターで４８時間培養後、培養上清を回収し創傷治癒関連の成長因子としてＶＥＧＦ、ＰＤＧＦ−ＡＢ、そしてＴＧＦ−β１の濃度測定を行った。濃度測定は市販のＥＬＩＳＡキット（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社）を用いた。
比較対照としては、培養前の培養液および不織布ろ過後に回収された血液を遠心して調製した血漿を用いた。結果を表５に示す。
〔試験例３〕：動物モデルを用いて創傷治癒効果の確認を以下のように行った。
２型糖尿病モデルマウス（Ｃ５７ＢＬ／ＫｓＪ−ｄｂ／ｄｂ Ｊｃｔ，日本クレア（株））の背部を全身麻酔下に剃毛した後，バイオプシー用の６ｍｍφパンチ（カイインダストリーズ社、ＢＰ−６０Ｆ）を用いて皮膚全層欠損処置を行った。マウスあたり３個の全層欠損処置を行った。
次に３個の全層欠損部位に各々実施例３にて血液処理を行ったポリ乳酸製不織布（即ち、血液細胞を表面に有する本発明の創傷治癒促進材）、血液処理を行っていないポリ乳酸製不織布、そして比較対照として何も貼付しない処置を行い、さらにこれらをポリウレタン製の創傷被覆材（『テガダーム』、３Ｍ社）で固定した。比較対照部位にはウレタン製創傷被覆材のみを貼付した。
２週間後にウレタンシートおよび創傷治癒促進材を剥がして傷の大きさを測定した。
創傷部位および定規をデジタルカメラで同時に測定し、得られた画像から画像解析ソフトＩｍａｇｅＪ（ｔｈｅ Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ）で解析することにより傷の面積を算出した。表６に全層欠損処置直後の創傷部位面積を１００としたときの２週間後の面積を算出しその治癒程度を比較した。
産業上の利用の可能性
本発明によれば、簡便な操作で短時間のうちに血液細胞を濃縮した創傷治癒促進材を調製できる。本発明の創傷治癒促進材は細胞の増殖を促進する効果を有することから、本発明の創傷治癒促進材を用いることによって創傷の治癒を促進することができる。[Example 1]: Preparation of wound healing promoter (human blood)
A sheet-like porous body having blood cells on its surface was prepared by the following method. Peripheral blood was collected from healthy volunteers using CPD (citrate-phosphate-dextrose) as an anticoagulant (blood: CPD = 400: 56, white blood cell count: 5,100 / μL, platelet count: 14. 4 × 10 4 / μL). A non-woven fabric made of polyethylene terephthalate (Asahi Kasei Co., Ltd., average fiber diameter 2.6 μm, thickness 0.38 mm, bulk density 0.27 g / cm 3 ) is punched into a 25 mm diameter, and two of these are stacked to form a column (Advantech, PP-25), and 5 mL of peripheral blood was allowed to flow through the column. After flowing blood, 10 ml of phosphate buffer was flowed through the column and washed to obtain a wound healing promoter containing a sheet-like porous body with blood cells attached to the surface.
The above operations were performed twice separately, but the time required for the treatment was 10 minutes or less.
The number of white blood cells and platelets contained in the blood before and after the treatment was measured with a hemocytometer.
The capture rate of leukocytes and platelets by the non-woven filter layer was as shown in Table 1 below.
A sheet-like porous body having blood cells on its surface was prepared by the following method. Peripheral blood was collected from healthy volunteers using heparin as an anticoagulant (blood: heparin = 100: 1, white blood cell count: 3,800 / μL, platelet count: 22.5 × 10 4 / μL). A non-woven fabric made of polyethylene terephthalate (Asahi Kasei Co., Ltd., average fiber diameter 1.1 μm, thickness 0.24 mm, bulk density 0.17 g / cm 3 ) is punched into a 25 mm diameter, and two of these are stacked to form a column (Advantech, PP-25), and 5 mL of peripheral blood was allowed to flow through the column. After flowing blood, 10 ml of phosphate buffer was flowed through the column and washed to obtain a wound healing promoter composed of a sheet-like porous body with blood cells attached to the surface.
The above operations were performed twice separately, but the time required for the treatment was 10 minutes or less.
The number of white blood cells and platelets contained in the blood before and after the treatment was measured with a hemocytometer.
The capture rate of leukocytes and platelets by the nonwoven fabric filter layer was as shown in Table 2 below.
A sheet-like porous body having blood cells on its surface was prepared by the following method.
Peripheral blood was collected from a type 2 diabetes model mouse (C57BL / KsJ-db / db Jct, Nippon Claire Co., Ltd.) using heparin as an anticoagulant (white blood cell count: 5,200 / μL, blood: heparin) = 100: 1). A polylactic acid non-woven fabric (Asahi Kasei Co., Ltd., average fiber diameter 1.14 μm, thickness 0.20 mm, bulk density 0.20 g / cm 3 ) is punched into a 13 mm diameter, and three of these are stacked to form a 5 mL syringe (Terumo) In this, 200 μL of mouse peripheral blood was allowed to flow. After flowing blood, 200 μL of phosphate buffer solution was flowed continuously for washing to obtain a wound healing promoter composed of a sheet-like porous body with blood cells attached to the surface.
The above operations were performed twice separately, but the time required for the treatment was within 2 minutes.
White blood cells contained in blood before and after treatment were measured with a hemocytometer.
The white blood cell capture rate by the nonwoven fabric filter layer was as shown in Table 3 below.
As a culture vessel, a multiple well culture plate (Corning, Transwell 3452) having inserts separated by a 3 μm pore size membrane was used.
Two non-woven fabrics (ie, wound healing promoting material of the present invention having blood cells attached to the surface) that were subjected to blood treatment in Example 1 are placed on the upper side separated by the membrane of the insert, and the human fetal lung derived from the lower side Normal diploid fibroblast cell line (HEL, human embryonic fibroblast). The seeding density of the cells was 1 × 10 5 cells / well each.
As specified by the manufacturer in Basal Medium Eagle (Sigma, B1522), 1 mM sodium pyruvate (ICN), 100-fold concentrated non-essential amino acid (ICN, Cat. # 1681049) was diluted 100-fold, and Glutamine was added to 2 mM. Further, fetal bovine serum (FCS, StemCell Technologies) and antibiotics such as penicillin and streptomycin were added to prepare a culture solution. The volume of the culture solution was 4 mL / well.
In addition, as a control, co-culture of a non-woven fabric that was not treated with blood and HEL was performed.
The above culture was cultured in an incubator at 5% CO 2 and 37 ° C. for 4 days, and the number of fibroblasts was measured. The results are shown in Table 4. For recovery of fibroblasts, a 0.25% Trypsin / EDTA solution (Gibco) was used.
[Test Example 2]: Growth Factor Production from Wound Healing Promoters Various growth factors produced from cells adhered by culturing the nonwoven wound healing promoter with blood cells prepared in Example 2 on the surface The concentration of was examined.
As a culture container, a culture dish (Asahi Techno Glass Co., Ltd.) having a diameter of 35 mm was used.
Two non-woven fabrics (that is, the wound healing promoter of the present invention having blood cells attached to the surface) subjected to blood treatment in Example 2 were placed.
Fetal bovine serum (FBS, Gibco) is added to a commercially available culture medium Dulbecco's Modified Eagle Medium (D-MEM, Gibco) to give a final concentration of 2%, and gentamicin as an antibiotic is added. Was made. The volume of the culture solution was 2 mL.
After culturing the above culture in an incubator at 5% CO 2 and 37 ° C. for 48 hours, the culture supernatant was collected, and the concentrations of VEGF, PDGF-AB, and TGF-β1 were measured as growth factors related to wound healing. . The concentration was measured using a commercially available ELISA kit (R & D Systems).
As a comparative control, plasma prepared by centrifuging the culture solution before culturing and blood collected after filtration of the nonwoven fabric were used. The results are shown in Table 5.
[Test Example 3] The wound healing effect was confirmed as follows using an animal model.
After shaving the back of a type 2 diabetes model mouse (C57BL / KsJ-db / db Jct, Nippon Claire Co., Ltd.) under general anesthesia, a 6 mmφ punch for biopsy (Kay Industries, BP-60F) was used. A full thickness skin defect treatment was performed. Three total thickness defect treatments were performed per mouse.
Next, the polylactic acid nonwoven fabric (that is, the wound healing promoting material of the present invention having blood cells on the surface) obtained by performing blood treatment in Example 3 on each of the three all-layer defect sites, and poly not subjected to blood treatment A lactic acid non-woven fabric and a treatment with nothing applied as a comparative control were performed, and these were fixed with a polyurethane wound dressing ("Tegaderm", 3M Company). Only the urethane wound dressing was applied to the comparative control part.
Two weeks later, the urethane sheet and the wound healing promoter were peeled off, and the size of the wound was measured.
The wound area and ruler were simultaneously measured with a digital camera, and the area of the wound was calculated by analyzing the obtained image with image analysis software ImageJ (the National Institutes of Health). In Table 6, the area after 2 weeks when the wound site area immediately after the all-layer defect treatment was defined as 100 was calculated, and the degree of healing was compared.
Industrial Applicability According to the present invention, it is possible to prepare a wound healing promoting material in which blood cells are concentrated in a short time by a simple operation. Since the wound healing promoter of the present invention has an effect of promoting cell proliferation, wound healing can be promoted by using the wound healing promoter of the present invention.
Claims (38)
Priority Applications (1)
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| JP2005506851A JP4847129B2 (en) | 2003-06-06 | 2004-06-07 | Wound healing promoter |
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| JP2003161887 | 2003-06-06 | ||
| JP2003161887 | 2003-06-06 | ||
| JP2005506851A JP4847129B2 (en) | 2003-06-06 | 2004-06-07 | Wound healing promoter |
| PCT/JP2004/008254 WO2004108146A1 (en) | 2003-06-06 | 2004-06-07 | Material promoting wound healing |
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| JPWO2004108146A1 JPWO2004108146A1 (en) | 2006-07-20 |
| JP4847129B2 true JP4847129B2 (en) | 2011-12-28 |
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| US (2) | US20060257456A1 (en) |
| EP (1) | EP1637145B1 (en) |
| JP (1) | JP4847129B2 (en) |
| KR (1) | KR20060028400A (en) |
| CN (1) | CN1798565A (en) |
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| WO (1) | WO2004108146A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20070113876A (en) * | 2006-05-26 | 2007-11-29 | 메디제네스(주) | A pharmaceutical composition for treatment of wounds containing blood plasma component |
| WO2010020247A1 (en) | 2008-08-22 | 2010-02-25 | Reapplix Aps | Multilayered blood product |
| CN107854717B (en) | 2009-01-27 | 2021-06-01 | 雷德德莱斯有限公司 | Wound dressing, method and apparatus for manufacturing wound dressing, and storage and use thereof |
| KR101422690B1 (en) * | 2009-02-27 | 2014-07-23 | (주)차바이오앤디오스텍 | Composition for skin regeneration by using medium or secretion of embryonic stem cell derived endothelial progenitor cells and use thereof |
| JP5646820B2 (en) * | 2009-04-16 | 2014-12-24 | 帝人株式会社 | Wound treatment material |
| US9243353B2 (en) | 2011-01-26 | 2016-01-26 | Asahi Kasei Fibers Corp. | Stent grafts |
| GB2489320A (en) | 2011-03-21 | 2012-09-26 | Univ Reading | Transport of cells by encapsulating in a hydrogel |
| KR101335176B1 (en) * | 2011-12-12 | 2013-11-29 | 테고사이언스 (주) | Wound Dressing |
| WO2017033734A1 (en) * | 2015-08-27 | 2017-03-02 | テルモ株式会社 | Blood component separation device and blood component separation method |
| EP3350312B1 (en) | 2015-09-15 | 2020-12-16 | Stellenbosch University | A method of culturing cells |
| WO2017176253A1 (en) * | 2016-04-05 | 2017-10-12 | General Electiric Comapny | Activated platelet composition with tunable growth factor level |
| KR102089712B1 (en) * | 2018-08-30 | 2020-03-16 | 메디칸(주) | Wound Dressing Comprising Non-Woven Fabric Sheet With Microscopic Pores for Blood Coagulation and Method of Manufacturing the Same |
| WO2022084478A1 (en) | 2020-10-21 | 2022-04-28 | Reapplix A/S | Blood product for preventing surgical adhesion |
| JP7248721B2 (en) * | 2021-02-17 | 2023-03-29 | ゼネラル・エレクトリック・カンパニイ | Activated platelet composition capable of modulating growth factor levels |
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Also Published As
| Publication number | Publication date |
|---|---|
| US20140335149A1 (en) | 2014-11-13 |
| JPWO2004108146A1 (en) | 2006-07-20 |
| US20060257456A1 (en) | 2006-11-16 |
| EP1637145B1 (en) | 2013-05-15 |
| EP1637145A1 (en) | 2006-03-22 |
| KR20060028400A (en) | 2006-03-29 |
| CN1798565A (en) | 2006-07-05 |
| EP1637145A4 (en) | 2009-05-13 |
| TW200505394A (en) | 2005-02-16 |
| WO2004108146A1 (en) | 2004-12-16 |
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